Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5' triphosphate (5'ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5'pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5'pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5'pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5'pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5'pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents

Techniques d'animation 3D et de synthèse d'images : programme d'études techniques, 574.B0

Titre de l'écran-titre (visionné le 22 août 2008)"Training sector 13 : communications and documentation"Traduction de: Techniques d'animation 3D et de synthèse d'images : programme d'études techniques, 574.B

Secteur de formation 13 : communications et documentation

Titre de l'écran-titre (visionné le 29 avril 2008)"Secteur de formation 13 : communications et documentation"Bibliogr

Systems Analysis of a RIG-I Agonist Inducing Broad Spectrum Inhibition of Virus Infectivity

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 59 triphosphate (59ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 59pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. [...] Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 59pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents

5′pppRNA treatment controls influenza-mediated pneumonia.

<p>Mice were treated with 5′pppRNA in complex in vivo-jetPEI on the day prior to influenza infection (Day −1) and the day of infection (Day 0). Lungs were collected on Day 3 and Day 8 post-infection and stained with hematoxylin and eosin (H&E). For each group, a representative picture showing (<b>A</b>) inflammation and tissue damage and (<b>B</b>) the extent of pneumonia is presented. (<b>C</b>) Inflammation, tissue damage and surface area affected by pneumonia were scored by a veterinary pathologist. Grade 1 = minimal; Grade 2 = modest, rare; Grade 3 = moderate, frequent; Grade 4 = severe, extensive.</p

5′pppRNA acts as a broad-spectrum antiviral agent.

<p>(<b>A</b>) A549 cells were transfected with 10 ng/ml 5′pppRNA 24 h prior to infection with VSVΔ51-GFP (MOI 0.1), Dengue virus (MOI 0.1), and Vaccinia-GFP virus (MOI 5), respectively. Percentage of infected cells was determined 24 h post-infection by flow cytometry analysis of GFP expression (VSV-GFP and Vaccinia-GFP) or intracellular staining of DENV E protein expression (Dengue virus). Data are from a representative experiment performed in triplicate ± SD. (<b>B</b>) Human PBMCs were transfected with 100 ng/ml 5′pppRNA 24 h prior to infection with dengue virus at an MOI of 5. At 24 h post-infection, the percentage of Dengue infected CD14+ and CD14− cells was evaluated by intracellular staining of DENV E protein expression by flow cytometry. Data are from a representative experiment performed in triplicate ± SD. (<b>C</b>) Human PBMCs from three different donors were transfected with 100 ng/ml 5′pppRNA prior to infection with Dengue virus at an MOI of 5. The percentage of Dengue infected cells in the CD14+ population was evaluated by intracellular staining of DENV E protein expression using flow cytometry. Data are from an experiment performed in triplicate on three different patients ± SD (<b>D</b>) CD4+ T cells isolated from human PBMCs and activated with anti-CD3 and anti-CD28 antibodies. Cells were incubated in the presence or absence of supernatant from 5′pppRNA-treated monocytes for 4 h and infected with HIV-GFP (MOI 0.1) for 48 h. The percentage of HIV infected, activated CD4+ T cells (GFP positive) was assessed by flow cytometry. (<b>E</b>) Huh7 and Huh7.5 were transfected with 5′pppRNA (10 ng/mL) and infected with HCV 24 h later. At 48 h post-infection, WCEs were collected and subjected to SDS PAGE and immunoblot to examine the expression of HCV viral protein NS3, IFIT1, and β-Actin.</p

5′pppRNA activates innate immunity and protects mice from lethal influenza infection <i>in vivo</i>.

<p>C57Bl/6 mice were injected intravenously with 25 ug of 5′pppRNA in complex with in vivo-jetPEI. (<b>A–B–C</b>) Mice were treated with 5′pppRNA on the day prior to influenza infection (500 PFU; Day −1) and the day of infection (Day 0). Percent survival (<b>A</b>) and percent weight loss (<b>B</b>) were monitored. (<b>C</b>) Lung viral titers were measured by plaque assay at the indicated day post-infection. Error bars represent SEM from six different animals. ND: not detected. (<b>D</b>) Repetitive administration of 5′pppRNA on the indicated days further decreases lung viral titers following infection with 500 PFU of influenza, as determined by plaque assay on Day 3 post-infection. Error bars represent SEM from five different animals. (<b>E</b>) Mice were infected with 50 PFU of influenza on Day 0. 5′pppRNA was administered prophylactically (Day −1, Day 0) or therapeutically (Day 1, Day 2) and lung viral titers were determined on Day 3. Error bars represent SEM from five different animals. (<b>F</b>) WT, TLR3−/−, MAVS−/− mice were treated with 5′pppRNA and serum IFNβ was quantified by ELISA at 6 h. Error bars represent SEM from three different animals. (<b>G</b>) WT and MAVS−/− mice were treated with 5′pppRNA and infected with influenza (500 PFU). Lungs were collected and homogenized on Day 1 and lung viral titers were determined by plaque assay. Error bars represent SEM from four different animals. (<b>H</b>) IFNα/βR−/− mice were either non-treated or treated with 5′pppRNA and infected with influenza (100 PFU). Survival was monitored for 18 days. Statistical analysis was performed by Student's t test (*, p≤0.05; **, p≤0.01; ***, p≤0.001; ns, not statistically significant).</p

5′pppRNA stimulates an antiviral and inflammatory response in lung epithelial A549 cells.

<p>(<b>A</b>) Schematic representation of VSV-derived 5′pppRNA and gel analysis. The 5′ppp-containing 67-mer RNA oligonucleotide is derived from the untranslated regions (UTRs) of VSV and the product of <i>in vitro</i> transcription runs as a single product degraded by RNase I. (<b>B</b>) 5′pppRNA or a homologous control RNA lacking a 5′-triphosphate end was mixed with Lipofectamine RNAiMax and transfected at different RNA concentrations (0.1–500 ng/ml) into A549 cells. At 8 h post treatment, whole cell extracts (WCEs) were prepared, resolved by SDS-page and analyzed by immunoblotting for IRF3 pSer396, IRF3, ISG56, NOXA, cleaved caspase 3, PARP and β-actin. Results are from a representative experiment; all immunoblots are from the same samples. (<b>C</b>) A549 cells were transfected with 10 ng/ml 5′pppRNA and WCEs were prepared at different times after transfection (0–48 h), subjected to SDS-PAGE and probed with antibodies for IRF3 pSer-396, IRF3, IRF7, STAT1 pTyr-701, STAT1, ISG56, RIG-I, IκBα pSer-32, IkBα and β-actin; all immunoblots are from the same samples. To detect IRF3 dimerization, WCEs were resolved by native-PAGE and analyzed by immunoblotting for IRF3. (<b>D</b>) ELISA was performed on cell culture supernatants to quantify the release of IFNβ and IFNα over time. Error bars represent SEM from two independent samples.</p